DE69432382T2 - N-Alkylacridancarboxylderivate, suitable for the detection of chemiluminescence - Google Patents

Abstract

N-alkylacridan carboxylic acid derivative compounds (I) are used to generate chemiluminescence by the action of a peroxidase enzyme and an oxidant. The compounds I are useful in assays of all types. <IMAGE>

Description

The present invention relates new N-alkylacridancarboxylic derivatives that generate light. The invention also concerns an improved method of chemically generating light (chemiluminescence) the effect of a peroxidase enzyme and an oxidizing agent, such as hydrogen peroxide with a group of N-alkylacridancarboxylic derivatives. The The invention also relates to a method for determining the extent of created this process Greatly increase chemiluminescence through the use of amplifiers. The invention also relates to the use of this method for detection of the peroxidase enzyme. The invention also relates to the application of this method to To detect hydrogen peroxide. Moreover, the invention relates the application of this method for detection and quantitative Detection of different biological molecules. The method can for Example applied to haptens, antigens and antibodies by an immunoassay procedure, Proteins by Western blotting, DNA and RNA by Southern and Northern blotting and nucleic acids by enzyme-linked nucleic acid probes demonstrated. The procedure can also be applied to the DNA in applications of DNA sequence analysis demonstrated. The method can also be used to To detect enzymes that produce hydrogen peroxide, such as glucose oxidase, Glucose-6-phosphate dehydrogenase, Galactose oxidase and the like.

The detection and quantification of biological molecules has hitherto been accompanied by the use of radioactively labeled reporter molecules excellent sensitivity. Recently, numerous non-radioactive Process designed to reduce the dangers and inconvenience avoid that caused by these materials. On enzyme-linked Analyte-based methods provide the best sensitivity there by the ability to convert the substrate catalytically, whereby a detectable change is caused, a gain is achieved. Substrates were developed that have a color, fluorescence or chemiluminescence, the latter being the most sensitive achieve.

Further improvements in sensitivity An assay extends the scope of chemiluminescence based method by allowing the detection of analytes, which are present in smaller quantities, or the time and / or amount the reagents is reduced for the implementation of the Assays are required. A method for improving speed and sensitivity of detection in an enzymatic chemiluminescent Assay consists of using substrates that have light with one higher Efficiency or over a longer one Create time.

Of the enzymes involved in enzymes Detection methods, such as immunoassays, the detection of oligonucleotides and nucleic acid hybridization methods, horseradish peroxidase is currently used most extensively used. To have the beneficial properties of this To use enzyme more efficiently in the analysis would be new chemiluminescent Substrates desired, which allow the detection of lesser amounts of the enzyme. In particular would be substrates beneficial, the stronger one Chemiluminescence either as higher maximum intensity or with a longer one Duration as compounds known in the art.

a. Oxidation of acridan

The oxidation of acridan by benzoyl peroxide in an aqueous solution produced chemiluminescence with a very low efficiency (⌀ _CL = 3 × 10 ^-7 ) and a mixture of products including acridine (S. Steenken, Photochem., Photobiol., 11, 279-283 (1970)). N-methylacridan is electrochemically oxidized to an N-methylacridinium ion (P. Hapiot, J. Moiroux, JM Saveant, J. Am. Chem. Soc., 112 (4), 1337-43 (1990), NW Koper, SA Jonker, JW Verhoeven Recl. Trav. Chim. Pays-Bas, 104 (11), 296-302 (1985)). Chemical oxidation of N-alkylacridan compounds was performed with a ferricyanide ion (A. Sinha, TC Bruice, J. Am. Chem. Soc., 106 (23), 7291-2 (1984)), certain quinones (AK Cotter P. Plank, JP Bergsma, R. Lahti, AA Quesnel, AG Parsons, Can. J. Chem., 62 (9), 1780-4 (1984)) and lithium nitrite (ON Chupakhin, IM Sosonkin, Al Matern, GN Strogov , Dokl. Akad. Nauk USSR, 250 (4), 875-7 (1980)). The oxidation of an N-alkylacridan derivative was carried out photochemically with or without a flavin compound as the co-oxidant (WR Knappe, J. Pharm. Sci., 67 (3), 318-20 (1978); GA Digenis, S. Shakshir, MA Miyamoto, NB Kostenbauer, J. Pharm. Sci., 65 (2), 247-51 (1976)).

Aryl and alkyl esters of 10-methylacridan-9-carboxylic acid undergo autooxidation to N-methylacridone in dipolar aprotic solvents under strongly basic conditions to produce chemiluminescence (F. McCapra, Accts. Chem. Res., 9 (6), 201-8 (1976)). The quantum yields of chemiluminescence ranged from 10 ^-5 to 0.1, and it was found to increase as the pK _S - value of the phenol or alcohol leaving group decreased. The quantum yields were significantly lower in an aqueous solution due to the competing non-luminescent decomposition of an intermediate. The addition of the cationic surfactant CTAB increased the apparent light output 130-fold by preventing the competing non-luminous reaction.

About the use of peroxidase or other enzymes to produce acridans or substituted acrids ne to oxidize, there are no reports. There are no reports of the development of chemiluminescence in the reaction of acridans or substituted acridans with peroxidase or any other enzymes.

b. chemiluminescent Oxidation of acridinium esters

The chemiluminescent oxidation of aliphatic and aromatic esters of N-alkylacridiniumcarboxylic acid by H ₂ O ₂ in an alkaline solution is a well-known reaction. The high quantum yield of chemiluminescence, which reaches 0.1, led to the development of derivatives with bonded reactive groups for binding to biological molecules. Numerous chemiluminescent immunoassays and assays with an oligonucleotide probe using acridinium ester markers have been reported.

The use of acridinium esters (AE), especially when they label a protein or oligonucleotide, has two disadvantages. The main problem is the limited hydrolytic Stability. Acridinium ester conjugates decay at or slightly above room temperature evenly. In dependence substitution of the leaving group may allow storage for extended storage required at -20 ° C his.

A second disadvantage of acridinium esters is the tendency to add nucleophiles such as water in the 9-position, thereby spontaneously generating a pseudobase intermediate which is non-luminescent and in a pH-dependent manner in a non-luminescent manner. shining process decomposes. In practice, the pH of solutions containing acridinium esters must first be reduced to reverse the formation of pseudobases and then increased in the presence of H ₂ O ₂ to produce light.

Recently also amides, thioesters and sulfonamides of N-alkylacridiniumcarboxylic acid have been prepared, and it was shown that she Emit light when oxidized under these conditions (T Kinkel, H. Lubbers, E. Schmidt, P. Molz, H. K. Skripczyk, J. Biolumin. Chemilumin., 4, 136-139 (1989); G. Zomer, J.F.C. Stravenuiter, Anal. Chim. Acta, 227, 11-19 (1989)). These modifications of the leaving group improve performance in terms of shelf life only partially.

A more fundamental restriction for use of acridinium esters as chemiluminescent labels is based on the fact that at the use as direct markers only up to about 10 molecules at one Protein or oligonucleotide can be bound. In conjunction with the Quantum yield in the generation of a photon (≤ 10%) can an acridinium ester labeled analyte will produce at most one photon. Another improvement of signal generation capability is not possible.

An attempt to reduce the number of in To increase an acridinium ester molecules associated with an analyte in an immunoassay was in the construction of an antibody-liposome conjugate, the liposome containing an indefinite number of AE (S.-J. Law, T. Miller, U. Piran, C. Klukas, S. Chang, J. Unger, J. Biolumin. Chemilumin., 4, 88-98 (1989)). Within a comparable assay, The directly marked AE used only a slight amplification of the Signals observed.

There is no known use of peroxidase or other enzyme associated with acridinium ester chemiluminescence.

c. chemiluminescent Detection of horseradish peroxidase

Amino-substituted cyclic acylhydrazides such as luminol and isoluminol react under basic conditions with H ₂ O ₂ and an enzyme catalyst in the form of peroxidase (such as horseradish peroxidase, HRP) with the emission of light. This reaction was used as the basis for analysis methods for detecting H ₂ O ₂ and the enzyme peroxidase. In conjunction with the use of luminol, various amplifiers have also been used to increase the intensity of the emitted light. These include D-luciferin (TP Whitehead, GN Thorpe, TJ Carter, C. Groucutt, LJ Kricka, Nature, 305, 158 (1983)) and p-iodophenol and p-phenylphenol (GH Thorpe, LJ Kricka, SB Mosely, TP Whitehead, Clin. Chem., 31, 1335 (1985)). At present, the only other chemiluminescent compound that is oxidized by the enzyme peroxidase and a peroxide is a hydroxy-substituted phthalhydrazide (Akhavan-Tafti, co-pending U.S. Patent Application No. 965,231, filed October 23, 1992).

The mechanism of the oxidation of phthalhydrazides by combining a peroxide and the enzyme peroxidase is very complex and continues to be the subject of intense debate. This problem has been diminished by the development of new chemiluminescent reactions catalyzed by peroxidase. Nevertheless, the horseradish peroxidase enzyme has found use in enzyme immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as a substrate (TP Whitehead, GH Thorpe, TJ Carter, C. Groucutt, LJ Kricka, Nature, 305 , 158 (1983)); GN Thorpe, LJ Kricka, SB Mosely, TP Whitehead, Clin. Chem., 1335 (1985)); GN Thorpe, SM Mosely, LJ Kricka, RA Stott, TP Whitehead, Anal. Chim. Acta, 170, 107 (1985) and JA Matthews, A. Batki, C. Hynds, LJ Kricka, Analy. Biochem., 151, 205 (1985)). There are commercially available kits for Available to associate HRP with improved chemiluminescent detection with luminol.

Synthetic peptide isoluminol derivatives, such as t-Boc-alanyl-alanyl-phenylalanyl iso-luminol amide, are substrates for the protease enzymes chymotrypsin, trypsin and thrombin. The transesterification of compounds of this type with the enzyme protease releases isoluminol, which can then react with the enzyme peroxidase and H ₂ O ₂ to produce chemiluminescence (BR Branchini, GM Salituro, in Bioluminescence and Chemiluminescence: Instrumental Applications, K.A. Van Dyke, ed., CRC Press, Boca Raton, Fla., Vol. 2, pp. 25-39, (1985)).

Urdea, U.S. Patent No. 5,132,204 a stable 1,2-dioxetane following each other Removal of the protecting groups by HRP and alkaline phosphatase from the phenol unit rapidly emitting a chemiluminescence decomposed. This double-protected However, compound is due to the slow thermal decomposition or Hydrolysis of the protecting group chemiluminescent even without an enzyme. No examples are given include the N-alkylacridancarboxylic derivatives.

It is therefore an object of present invention, a method and N-alkylacridancarboxylic derivatives for use in the generation of chemiluminescence by the action of Enzyme peroxidase for indicate the detection of biological materials and compounds. It is also an object of the present invention to provide a method and a kit containing the N-alkylacridancarboxylic derivatives in a solution or on surfaces, like membranes, use, for the use in generating a chemiluminescence by the Effect of the enzyme peroxidase for to provide detection of peroxidase enzymes and enzyme conjugates. In addition It is an object of the present invention, a method and a Kit, the N-alkylacridancarboxylic derivatives use, for the use in generating a chemiluminescence by the Effect of the enzyme peroxidase for use in nucleic acid assays in a solution and on surfaces specify. In addition It is an object of the present invention to provide a method and a kit, the N-alkylacridancarboxylic derivatives use, for the use in generating a chemiluminescence by the Effect of the enzyme peroxidase for the detection of proteins in Western blots and of DNA in Southern blots and other DNA hybridization assays specify.

Show it:

1 a graph showing a comparison of the light emission curves of a series of five Acridanverbindungen invention. A 200 μl volume of reagent composition containing 0.1 mM of acridan compound 1a-e in 0.1 M phosphate buffer, pH 8.9, 0.015% (6.2 mM) H ₂ O ₂ , 2.25 mM p Iodophenol, 0.5% (w / w) Tween 20 1 mM EDTA was reacted with 1 x 10 ^-13 moles of horseradish peroxidase.

2A . 2 B and 2C a set of graphs showing the relationship between a background corrected signal and the amount of HRP using solutions containing 0.05 mM 1b, 0.5% Tween 20.1 x 10 ^-3 M EDTA in pH = 8.0, 0.1 M Tris buffer. These three graphs show the influence of the H ₂ O ₂ concentration and the reaction time on the sensitivity and linearity of the detection.

3A . 3B and 3C a set of graphs showing the relationship between a background corrected signal and the amount of HRP using solutions containing 0.05 mM 1b, 0.5% Tween 20.1 x 10 ^-3 M EDTA in pH = 8.0, 0.1 M Tris buffer. These three graphs show the influence of the NaBO ₃ concentration and the reaction time on the sensitivity and linearity of the detection.

4 Figure 4 is a graph showing the relationship between the background corrected signal and the amount of HRP using solutions containing p-iodophenol (0-4.5 mM), 0.5% Tween 20.1 × 10 ^-3 M EDTA in pH = 8.0, 0.1 M Tris buffer and 7 x 10 ^-16 moles of HRP, after a reaction time of 15 minutes at 37 ° C. The concentrations of 1b and H ₂ O ₂ are as in 4 specified.

5 a graphic representation of the results at 25 and 37 ° C. The curves of light intensity versus time shown by treatment of 200 μl of a formulation containing 0.05 mM of compound b 0.2 mM H ₂ O ₂ , 0.5 mM p-iodophenol, 0.5% (Fig. W / w) Tween 20 and 1 mM EDTA in 0.1 M Tris buffer, pH = 8.0, incubated at 25 or 37 ° C, with 10 μl of a solution containing 7 x 10 ^-6 moles of horseradish peroxidase. The chemiluminescence intensity reaches the maximum faster at 37 ° C.

6 a log-log curve showing the linearity of detection of HRP with a reagent composition of the present invention as compared to a commercial optimized reagent containing luminol. The reagent of the invention comprises 40 μl of a solution containing 1b (0.05 mM), p-iodophenol (2.25 mM), N ₂ O ₂ (0.2 mM), Tween 20 (0.5%), EDTA (1 mM) in 0.1 M Tris, pH = 8.0. For comparison, 40 .mu.l of the acridan reagent and the luminol reagent were incubated separately at 37.degree. C. and reacted with different amounts of HRP. The graph compares the signal-to-background ratios at 5 min. The reagent containing 1b may show a higher sensitivity in detection than the reagent luminol (compare log (S-B) / B = 0). This improved sensitivity is evident by comparing log (moles of HRP) for each reagent at the same value of log [(S-B) / B] to zero.

7A and 7B the result of analysis of Western blots of human transferrin on nitrocellulose in chemiluminescent detection using fractionated goat anti-human transferrin serum, a rabbit anti-goat IgG peroxidase conjugate, NaBO ₃ and 1b , The human transferrin charged in each gap was (1) 5,000 pg, (2) 1,000 pg, (3) 200 pg. (4) 50 pg and (5) 20 pg. After a 20 minute incubation, the blots were exposed to X-ray film X-OMAT AR (Kodak, Rochester, NY) for 7 seconds (FIG. 7A) or to X-ray film OMC after a 40 minute incubation for 30 seconds (FIG. 7B).

wobei R₁ aus Alkyl-, Heteroalkyl- und Aralkylgruppen ausgewählt ist und R₃ und R₄ aus Aryl-, Alkyl-, Heteroalkyl- und Aralkylgruppen ausgewählt sind.The present invention relates to an acridan of the formula

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups and R ₃ and R _{4 are selected} from aryl, alkyl, heteroalkyl and aralkyl groups.

umfasst, worin R₁ aus Alkyl-, Heteroalkyl- und Aralkylgruppen ausgewählt ist, R₅ und R₆ aus der Gruppe ausgewählt sind, die aus Wasserstoff und nicht störenden Substituenten besteht, und Y eine Abgangsgruppe ist, die die Erzeugung von Licht aus dem Acridan durch die Umsetrung mit einem Peroxid und einer Peroxidase ermöglicht.The present invention relates to a method of producing chemiluminescence which comprises reacting a peroxide compound and a peroxidase with an acridan of the formula

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which is the generation of light from the acridan by the Umsetrung with a peroxide and a peroxidase allows.

The present invention relates also a Reagenzzusammensetrung, in the presence of peroxidase Generates light and has the following:

• (a) an acridan of the formula:
  
  wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Permits permeation with a peroxide and a peroxidase;
• (b) optionally, a phenolic compound that promotes light generation amplified from the acridan;
• (c) a peroxide compound which is involved in the conversion of the acridan involved with the peroxidase;
• (d) a chelating agent which prevents the peroxide compound from reacting, before the peroxidase is added to this composition; and
• (e) a nonionic surfactant.

wobei R₁ aus Alkyl-, Heteroalkyl- und Aralkylgruppen ausgewählt ist, R₅ und R₆ aus der Gruppe ausgewählt sind, die aus Wasserstoff und nicht störenden Substituenten besteht, und Y eine Abgangsgruppe ist, die die Erzeugung von Licht aus dem Acridan durch die Umsetrung mit einem Peroxid und einer Peroxidase ermöglicht.The present invention also relates to an improved method of detecting an analyte in an assay method by a chemiluminescent reaction, the improvement comprising reacting the acridan with a peroxide and a peroxidase to produce light for detecting the analyte, the acridan having the formula: :

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Umsetrung with a peroxide and a peroxidase allows.

The present invention relates also an improved method for detecting an analyte in a Assay method by a chemiluminescent reaction, wherein the Improvement comprises:

• (a) providing a reagent composition which produces light in the presence of peroxidase and which comprises an acridan of the formula
  
  wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Permits permeation with a peroxide and a peroxidase; optionally a phenolic compound which enhances the production of light from the acridan; a peroxide compound involved in the conversion of the acridan with the peroxidase; a chelating agent which prevents the peroxide compound from reacting before the peroxidase is added to this composition; and a nonionic surfactant; and
• (b) adding peroxidase to the reagent composition, so that light is produced for the detection of the analyte.

The present invention relates also a kit for the detection of an analyte in an assay by a chemiluminescent A reaction whereby light is generated having in separate containers:

• (a) an acridan of the formula:
  
  wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Reaction with a peroxide and a peroxidase allows;
• (b) the enzyme peroxidase, the light being in the assay method by the reaction of the reagent composition with the peroxidase is detected.

The present invention relates also a kit for detecting an analyte in an assay procedure a chemiluminescent reaction, which generates light, that in separate containers comprising:

• (a) a reagent composition, the components of which may be in a single or multiple containers, which produces light in the presence of peroxidase, comprising: an acridan of the formula
  
  wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Permits permeation with a peroxide and a peroxidase; optionally a phenolic compound which enhances the production of light from the acridan; a peroxide compound involved in the conversion of the acridan with the peroxidase; a chelating agent which prevents the peroxide compound from reacting before the peroxidase is added to this composition; and a nonionic surfactant; and
• (b) the enzyme peroxidase, wherein the light in the detection method by reacting the reagent composition with the peroxidase is detected.

wobei R₁ aus Alkyl-, Heteroalkyl- und Aralkylgruppen ausgewählt ist, R₅ und R₆ aus der Gruppe ausgewählt sind, die aus Wasserstoff und nicht störenden Substituenten besteht, und Y eine Abgangsgruppe ist, die die Erzeugung von Licht aus dem Acridan durch die Umsetzung mit einem Peroxid und einer Peroxidase ermöglicht.The present invention also relates to an improved method of detecting hydrogen peroxide in an assay method by a chemiluminescent reaction, the improvement comprising reacting hydrogen peroxide and peroxidase with an acridan of the formula:

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which prevents the generation of light from the acridan by the Implementation with a peroxide and a peroxidase allows.

The preferred compounds according to the invention are the following compounds 1a to 1e:

The present invention includes a method for producing chemiluminescence from the oxidation of N-alkylacridancarboxylic acid derivatives (I) by the action of the enzyme peroxidase, a peroxide compound and enhancers. The invention also relates to the use of this method to detect the enzyme peroxidase with high sensitivity. Moreover, the invention relates to the use of the method for the detection and quantification of various biological molecules bound to this enzyme by chemical bonds or physical interactions. The intensity of the resulting chemiluminescence provides a direct characteristic of the amount of labeled organic or biological molecule. The method can be used, for example, to detect haptens, antigens and antibodies by an immunoassay method, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, and nucleic acids by enzyme-linked nucleic acid probes. The method may also serve to analyze the DNA in DNA sequence analysis se applications. The method can be used to detect hydrogen peroxide produced by enzymes such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase, galactose-6-phosphate dehydrogenase and amino acid oxidase. The method can therefore also serve as a means to detect the aforementioned enzymes which produce hydrogen peroxide.

The Umsetrung invention can in a Solution, like an aqueous buffer, or on the surface a solid support, like beads, a tube, a plate with microwells or a membrane, as is well known to those skilled in the art. If the evidence is up a membrane, this membrane may optionally in Kit be provided.

Detection of chemiluminescence by the oxidation of an N-alkylacridancarboxylic acid derivative by hydrogen peroxide catalyzed by enzyme peroxidase can be made with good sensitivity. Enhancement of this conversion by introducing chemiluminescent enhancing substances has enabled the measurement of chemiluminescence with even lower levels of the enzyme peroxidase. Coupling this enzyme to a biological molecule of interest then allows detection of this biological molecule with high sensitivity. The preferred amounts of the various components of the composition of the invention are listed in Table I. TABLE I Acridan I 0.01-10mM Phenol amplifier 0.001-10 mM Surfactant 0.01-5% peroxide 0.01-10mM EDTA 0.01-5 mM

The general reaction used for the production of light with N-alkylacridancarboxylic acid derivatives (I) is as follows:

An unexpected finding of the present invention is that N-alkylacridancarboxylic acid derivatives (I) are oxidized by the enzymes peroxidase in the presence of a peroxide to produce chemiluminescence. The chemiluminescence probably arises from the excited state of the N-alkylacridone. N-alkylacridanecarboxylic acid derivatives which have been found to react include esters, especially aromatic esters, and sulfonamides. There are other derivatives which provide a leaving group whose Säurekonjugat has a _pK a value of less than about 16 such as thioesters and alkyl esters are contemplated. N-alkylacridanecarboxylic acid derivatives having substituents on the aromatic groups of the acridan compound can generate light in the same manner. Non-interfering substituents, such as an alkyl, alkoxy, aralkyl, heteroalkyl, and carbon- and / or heteroatom-containing groups which provide a reactive group for the compound with other molecules or which provide better water solubility, may be present in one or both of the aromatic groups Be included in wrestling.

It was also found that this Introduce certain substituted phenolic compounds in combination with nonionic surfactant Agents in the reaction mixture in the presence of the added Peroxidase and the added peroxide chemiluminescence produced strengthened. Phenol compounds in which an increase in the extent of Chemiluminescence was found in the transposition of N-alkylacridanecarboxylic acid derivatives (I) produced with a peroxide compound and the enzyme peroxidase will close the following are but not limited to: p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, 2-naphthol and 6-bromo-2-naphthol. It is significant that the Phenol amplifier in the promotion the implementation of Hydroxylarylacridanestern are effective, the self contain a phenol substituent.

A key feature in the development of the most sensitive Detection systems consists in providing the possible most Signal through a reinforcement, such as by using an enzyme as detectable Substance, wherein the background signal in relation to the signal to be measured at one possible low value is maintained. Consequently, additives are used the generation of chemiluminescence from the conversion of hydrogen peroxide and N-alkylacridanecarboxylic acid derivatives (I) without suppressing the peroxidase enzymes to the applicability to improve this invention. It has also been found that surfactant Agent, such as nonionic surfactant Means for improving the applicability of the present invention by providing a better signal-to-background ratio. The improvement results from minimizing the background chemiluminescence without the added peroxidase, possibly due to a slowing of the auto - oxidative decomposition of the Acridan derivative.

Another aspect of the invention consists in the use of leaving groups of hydroxy-substituted Aryl esters. The further hydroxy substituent provides better stability for the Ester function in comparison with other functional groups, especially at pHs where phenol is substantially ionized. Also subject the hydroxyarylacridan esters under the action of the enzyme peroxidase and the peroxide of a fast and effective chemiluminescent Reaction.

The preferred system includes a solution in an aqueous buffer, which contains: 1) a phenolic enhancer, 2) a peroxide compound, wherein the peroxide compound is hydrogen peroxide, Urea peroxide or a perborate salt, 3) 4'-hydroxyphenyl-10-methylacridan-9-carboxylate, 4) a means for forming a cationic complex, wherein the Funds selected from the group which may consist of chelating agents, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic (DTPA) or ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) and their salts, and 5) a surfactant such as anionic surface-active Means sodium dodecyl sulfate (SDS) or preferably a nonionic surfactant Agents such as polyoxyethylenated alkylphenols, polyoxyethylenated Alcohols, polyoxyethylenated ethers, polyoxyethylenated sorbitol esters and the same.

In a preferred method of practicing the present invention, an aqueous buffer solution having a pH in the range of 8-10, the 4'-hydroxyphenyl-l0-methylacridan-9-carboxylate with a final concentration of about 0.01 to 1 × 10 ^{- 4} M, a phenol compound such as containing p-phenylphenol at a final concentration of about 0.01 to 1 × 10 ^-6 M and a nonionic surfactant at a final concentration of about 5 to 0.01% (V./V.) mixed with a second solution in water or an aqueous buffer containing a peroxide source, such as hydrogen peroxide or, preferably, a perborate salt, and a cationic complex forming agent, such as EDTA, having a final concentration of about 1 × 10 ^-3 to 1 × 10 -4 ^{. 5} M, whereby the solution of the detection reagent is generated. This solution is contacted with the enzyme peroxidase, which may either be in solution or attached to a solid support. Optimal concentrations of the reagents are easily determined individually for each composition. In particular, the concentration of the amplifier is optimized for each different amplifier used to produce the maximum amplification of the light emission.

Significant advantages of N-alkylacridancarboxylic acid derivatives (I) and the compositions according to the invention containing them in the higher Sensitivity for the detection of the enzyme peroxidase. Comparative experiments show a 10-fold reduction in the detection limit of HRP when the reagent composition according to the invention is used, compared with the reinforced luminol system. A second Advantage is the possible wide dynamic range the peroxidase concentration. Another advantage of N-alkylacridancarboxylic acid derivatives (I) is their thermal and photochemical stability and simple Cleaning. Most commonly known chemiluminescent Substrates or peroxidase enzymes known in the art are, cyclic aminoaryldiacylhydrazides, such as luminol, and these containing compositions, easily decompose in room light, resulting in a loss of sensitivity and a bad one Reproducibility leads, when used in chemiluminescent detection schemes (Y. Omote, H. Yamamoto, N. Sugiyama, Chem. Commun., 914 (1970)). cyclic Aminoaryldiacylhydrazides are difficult to produce and in one high purity state and must either be protected from light or be cleaned immediately before use (R.A.W. Stott, L.J. Kricka, Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al., Eds., Pp. 237-240 (1987)). Another Advantage of the use of certain N-alkylacridanecarboxylic acid derivatives (I) in comparison with conventional Connections exists in the longer Chemilumineszenzdauer. A longer one Duration simplifies the measurement by providing the required exact time Control of the reaction is omitted and increased the sensitivity of detection when based on a film Detection methods are applied.

EXAMPLES

1. Synthesis of the acridan derivative 1a

Phenyl acridine-9-carboxylate

Acridine-9-carboxylic acid (1 g, 4.1 mmol) was suspended in thionyl chloride (5 ml) and the reaction mixture was refluxed for 3 hours. The solvent was removed under reduced pressure leaving a yellow solid which was dissolved under argon in methylene chloride and pyridine (350 μl). This solution was cooled in an ice-bath and a solution of phenol (0.78 g, 8.2 mmol) in methylene chloride was added dropwise. The reaction mixture was stirred at room temperature overnight. After evaporation of the solvent, the residue was redissolved in ethyl acetate and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give a crude material which was chromatographed on silica gel (30% ethyl acetate / hexane) to give the pure product as a yellow solid.
¹ H NMR (CDCl ₃₎ δ 7.35 to 7.57 (m, 5H), 7.63 to 8.37 (m, 8H).

Phenyl-l0-methyl-acridinium-9-carboxylate trifluoromethanesulfonate

Phenylacridine-9-carboxylate (530mg, 1.7mmol) was dissolved in methylene chloride (5mL) under argon and methyl trifluoromethanesulfonate (1mL, 8.8mmol) was added. The solution was stirred at room temperature overnight to give a thick yellow precipitate. This precipitate was filtered, washed with ether and dried to give the product as yellow crystals.
¹ N NMR (acetone-d ₆ ) δ 5.22 (s, 3H), 7.47-7.71 (m, 5H), 8.23-9.07 (m, 8H).

Phenyl-l0-methylacridan-9-carboxylate (1a)

Phenyl-10-methylacridinium-9-carboxylate trifluoromethanesulfonate (10 mg, 0.022 mmol) was suspended in absolute ethanol (10 ml) and the mixture was refluxed for 15 minutes to give a clear solution. To the solution was added ammonium chloride (88 mg, 1.6 mmol) in portions followed by zinc (108 mg, 106 mmol). The addition of zinc caused the yellow color of the solution to disappear immediately. The colorless solution was refluxed for 2 hours. The TLC of the reaction mixture showed complete conversion to a non-polar material. The solution was filtered and the precipitate was washed with ethanol (3 x 20 ml). The filtrate was concentrated to give an off-white solid which was redissolved in methylene chloride and washed with water (2 x 15 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative TLC using 30% ethyl acetate: hexane. The pure product was obtained as an off-white solid.
¹ H NMR _(CDCl3) δ 3.38 (s, 3H), 5.16 (s, 1H), 6.89 to 7.37 (m, 13H)
¹³ C NMR _(CDCl3) δ 33.29, 49.72, 112.93, 120.19, 121.36, 125.73, 128.67, 129.16, 129.26, 142.37, 151, 04, 170,22.

2. Synthesis of the acridan derivative 1b

4- (tert-butyldimethylsilyloxy) phenol

To a solution of hydroquinone (1.0 g, 0.9 mmol) and tert -butyldimethylsilyl chloride (1.4 g, 0.9 mmol) in 5 mL of dry DMF was gradually added imidazole (1.2 g, 1.8 mmol ), and the solution was stirred for 1 hour. TLC analysis (silica gel, 20% ethyl acetate / hexane) showed the completion of the reaction. The solution was poured into 25 ml of water and extracted with 3 × 25 ml of ether. The mixed ether solutions were dried over anhydrous MgSO ₄ . Evaporation of the solvent gave an oil which was chromatographed on silica gel with 20% ethyl acetate / hexane to give the product as a white solid with a yield of 70%.
¹ H NMR (CDCl ₃ ) δ 0.145 (s, 6H), 0.956 (s, 9H), 4.47 (bs, 1H), 6.68 (s, 4H)
¹³ C NMR (CDCl ₃ ) δ -4.48, 18.21, 25.74, 115.93, 120.55, 120.81, 149.78.

4 '(tert-butyldimethylsilyloxy) phenyl acridine-9-carboxylate

Acridine-9-carboxylic acid (800 mg, 3.8 mmol) was suspended in thionyl chloride (5 ml) and the reaction mixture was refluxed for 3 hours. The solvent was released at reduced pressure to give a yellow solid which was dissolved under argon in methylene chloride and pyridine (1.5 ml). This solution was cooled in an ice-bath and a solution of 4- (tert-butyldimethylsilyloxy) phenyl (1.2 g, 5.3 mmol) in methylene chloride was added dropwise. The reaction mixture was stirred at room temperature overnight. The solution was diluted with more methylene chloride and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give a crude material which was chromatographed on silica gel (25% ethyl acetate / hexane) to give the pure product as a yellow solid.
¹ H NMR (CDCl ₃ ) δ 0.257 (s, 6H), 1.026 (s, 9H), 6.96-7.34 (dd, 4H), 7.64-8.34 (m, 8H)
¹³ C NMR (CDCl ₃ ) δ -4.38, 18.27, 25.72, 120.96, 122.19, 122.45, 127.52, 127.96, 130.57, 144.47, 148 , 56, 154, 03, 166, 15, 204, 64.

4'-hydroxyphenyl-l0-methyl-acridinium-9-carboxylate

4 '(tert -Butyldimethylsilyloxy) phenylacridine-9-carboxylate (410 mg, 0.98 mmol) was dissolved in methylene chloride (5 mL) under argon and methyl trifluoromethanesulfonate (558 μL, 4.9 mmol) was added. The yellow solution turned dark brown. After the solution was stirred for 2 hours at room temperature, a precipitate formed and the color of the solution changed from black to yellow. The solution was stirred at room temperature overnight to give a thick yellow precipitate. This precipitate was filtered, washed with ether and dried to give the product as yellow crystals.
¹ H NMR (acetone-d ₆ ) δ 5.24 (s, 3H), 7.02-7.53 (dd, 4H), 8.26-9.07 (m, 8H).

4'-hydroxyphenyl-l0-methylacridan-9-carboxylate (1b)

4'-Hydroxyphenyl-10-methylacridinium-9-carboxylate trifluoromethanesulfonate (500mg, 1mmol) was suspended in absolute ethanol (70ml) and the solution was heated at reflux for 30 minutes. Ammonium chloride (5.6 g, 0.104 mol) was added in portions to the heterogeneous solution followed by zinc (6.8 g, 0.104 mol). The yellow color of the solution disappeared immediately after the addition of zinc. The colorless solution was refluxed for 3 hours. The TLC of the reaction mixture showed complete conversion to a non-polar material. The solution was filtered and the precipitate was washed with ethanol (3 × 30 ml). The solution was concentrated to give an off-white solid which was redissolved in methylene chloride and washed with water (2 x 30 mL). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the product as an off-white solid.
¹ N NMR _(CDCl3) δ 3.42 (s, 3H), 4.69 (s, 1H), 5.16 (s, 1H), 6.65-6.78 (dd, 4H), 6, 97-7.37 (m, 8H).

3. Synthesis of the acridan derivative 1c

3-Lert.-butyldimethylsilyloxy) phenol

To a solution of resorcinol (1.0 g, 0.9 mmol) and tert-butyldimethylsilyl chloride (1.4 g, 0.9 mmol) in 5 mL of dry DMF was added gradually imidazole (1.2 g, 1.8 mmol ), and the solution was stirred for 1 hour. TLC analysis (silica gel, 20% ethyl acetate / hexane) showed the completion of the reaction. The solution was poured into 25 ml of water and extracted with 3 × 25 ml of ether. The mixed ether solutions were dried over anhydrous MgSO ₄ . Evaporation of the solvent gave an oil which was chromatographed on silica gel using 20% ethyl acetate / hexane to give the product as a white solid with a yield of 70%.
¹ H NMR (CDCl ₃ ) δ 0.199 (s, 6H), 0.983 (s, 9H), 6.39-7.09 (m, 4H).

3 '- (tert-butyldimethylsilyloxy) phenyl acridine-9-carboxylate

Acridine-9-carboxylic acid (700 mg, 3.3 mmol) was suspended in thionyl chloride (5 ml) and the reaction mixture was refluxed for 3 hours. The solvent was removed under reduced pressure to give a yellow solid which was dissolved under argon in methylene chloride and pyridine (355 μl). This solution was cooled in an ice-bath and a solution of 4- (tert-butyldimethylsilyloxy) phenol (400 mg, 5.3 mol) in methylene chloride was added dropwise. The reaction mixture was stirred at room temperature overnight. After evaporation of the solvent, the residue was redissolved in ethyl acetate and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give a crude material which was chromatographed on silica gel (30% ethyl acetate / hexane) to give the pure product as an off-white solid.
¹ H NMR _(CDCl3) δ 0.273 (s, 6H), 1.026 (s, 9H), 6.84 to 8.36 (m, 12H).

3'-Hydroxyphenyl-l0-methyl-acridinium-9-carboxylate trifluoromethanesulfonate

3 '- (tert-Butyldimethylsilyloxy) phenylacridine-9-carboxylate (110 mg, 0.025 mmol) was dissolved in methylene chloride (5 ml) under argon and methyl trifluoromethanesulfonate (145 μl, 1.2 mmol) was added. The solution was stirred at room temperature overnight to give a thick yellow precipitate. This precipitate was filtered, washed with chloroform and dried to give the product as yellow crystals.
¹ H NMR (acetone-d ₆ ) δ 5.22 (s, 3H), 6.91-7.42 (m, 4H), 8.22-9.05 (m, 12H), 8.95 (bs , 1H).

3'-Nydroxyphenyl-l0-methylacridan-9-carboxylate (1c)

3'-Hydroxyphenyl-10-methylacridinium-9-carboxylate trifluoromethanesulfonate (500mg, 1mmol) was suspended in absolute methanol (70ml) and heated at reflux for 30 minutes. Ammonium chloride (5.6 g, 0.104 mol) was added in portions to the heterogeneous mixture followed by zinc (6.8 g, 0.104 mol). The yellow color of the solution disappeared immediately after the addition of zinc. The colorless solution was refluxed for 3 hours. The TLC of the reaction mixture showed complete conversion to a non-polar material. The reaction mixture was filtered and the precipitate was washed with ethanol (3 × 30 ml). The filtrate was concentrated to give an off-white solid which was redissolved in methylene chloride and washed with water (2 × 30 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the product as an off-white solid.
¹ N NMR _(CDCl3) δ 3.42 (s, 3H), 4.85 (s, 1H), 5.17 (s, 1H), 6.37 to 7.37 (m, 12H).

4. Synthesis of the acridan derivative 1d

6- (tert-butyldimethylsilyloxy) -2-naphthol

To a solution of 2,6-dihydroxynaphthalene (1.4 g, 8.7 mmol) and tert-butyldimethylsilyl chloride (1.31 g, 8.7 mmol) in 5 mL of dry DMF was added gradually imidazole (1.2 g, 17 mmol), and the solution was stirred for 1 hour. The solution was poured into 25 ml of water and extracted with 3 × 25 ml of ether. The mixed ether solutions were dried over anhydrous MgSO ₄ . Evaporation of the solvent gave a solid which was dissolved in hexane and filtered to remove the unreacted starting material. The crude material was chromatographed on silica gel using 20% ethyl acetate / hexane to give the product as a white solid with a yield of 75%.
¹ N NMR (CDCl ₃ ) δ 0.219 (s, 6H), 1.002 (s, 9H), 4.81 (s, 1H), 7.01-7.60 (m, 6H) ¹³ C NMR (CDCl ₃ ) δ -4.17, 18.42, 25.92, 109.92, 115.28, 118.32, 122.80, 127.91, 128.62, 130.04, 130.38, 151.77, 151.92.

6 '- (tert-butyldimethylsilyloxy) acridine-9-carboxylate

Acridine-9-carboxylic acid (500 mg, 2.2 mmol) was suspended in thionyl chloride (5 ml) and the reaction mixture was refluxed for 3 hours. The solvent was removed under reduced pressure to give a yellow solid which was dissolved under argon in methylene chloride and pyridine (100 μl). This solution was cooled in an ice-bath and a solution of 6- (tert-butyldimethylsilyloxy) -2-naphthol (735 mg, 2.6 mmol) in methylene chloride was added dropwise. The reaction mixture was stirred at room temperature overnight. The solution was concentrated to give a solid which was dissolved in ethyl acetate and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give a crude material which was chromatographed on silica gel (25% ethyl acetate / hexane) to give the pure product as a yellow solid (65%).
¹ H NMR _(CDCl3) δ 0.278 (s, 6H), 1.044 (s, 9H), 7.16 to 8.34 (m, 14H)
¹³ C NMR (CDCl ₃ ) δ -4.25, 18.34, 25.78, 115.14, 118.47, 120.95, 122.49, 123.33, 124.98, 127.56, 128 , 58, 129, 22, 129, 40, 130, 17, 133, 15, 135, 97, 146, 62, 148, 75, 153.92, 166, 32.

6'-hydroxynaphthyl-l0-methyl-acridinium-9-carboxylate trifluoromethanesulfonate

6 '- (tert -Butyldimethylsilyloxy) naphthylacridine-9-carboxylate (500mg, 1mmol) was dissolved in methylene chloride (5ml) under argon and methyl trifluoromethanesulfonate (1.2ml, 10mmol) was added. There was a dark orange solution. The solution was stirred at room temperature overnight to give a thick yellow precipitate. This precipitate was filtered, washed with ether and dried to give the product as orange crystals.
¹ H NMR (acetone-d ₆ ) δ 5.25 (s, 3H), 7.27-9.09 (m, 15H), 8.90 (s, 1H).

6'-hydroxynaphthyl-l0-methylacridan-9-carboxylate (1d)

6'-Hydroxynaphthyl-10-methylacridinium-9-carboxylate trifluoromethanesulfonate (350 mg, 0.66 mmol) was suspended in absolute ethanol (30 mL) and heated at reflux for 15 minutes. To the mixture was added ammonium chloride (3.5 g, 66 mmol) in portions followed by zinc (4.3 g, 66 mmol). The yellow color of the solution disappeared immediately after the addition of zinc. The colorless solution was refluxed for 4 hours. The TLC of the reaction mixture showed complete conversion to a non-polar material. The solution was filtered and the precipitate was washed with ethanol (3 × 30 ml). The ethanol was evaporated to give an off-white solid which was redissolved in ethyl acetate and washed with water (2 x 30 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was chromatographed on silica gel (30% ethyl acetate / hexane) to give the pure product as an off-white solid.
¹ N NMR (acetone-d ₆ ) δ 3.38 (s, 3H), 5.34 (s, 1H), 6.94-7.70 (m, 14H), 8.70 (bs, 1H)
¹³ C NMR (CDCl ₃ ) δ 33.53, 50.02, 118.97, 120.02, 121.46, 122.09, 128.21, 129.35, 129.95, 130.79, 133, 79, 143, 36, 147, 56, 156, 11, 171, 05.

5. Synthesis of the acridan derivative 1e

N- (phenyl) -p-toluenesulfonamide

Aniline (1.86 g, 0.02 mol) was dissolved in methylene chloride under nitrogen and triethylamine (3.8 mL, 0.02 mol) was added. This solution was cooled in an ice bath and a solution of p-toluenesulfonyl chloride (3.8 g, 0.02 mol) was added dropwise via syringe. After this solution was stirred at room temperature for 4 hours, TLC analysis (silica gel, 20% ethyl acetate / hexane) showed the completion of the reaction. The reaction mixture was poured into ether and the precipitate was filtered. The ether layer was washed with water, dried over MgSO ₄ and concentrated to give an oily material. This crude product was chromatographed over silica using 35% ethyl acetate and hexane to give a solid which was also recrystallized in methylene chloride / hexane, mp 104 ° C.
¹ H NMR _(CDCl3) δ 2.36 (s, 3H), 7.07 to 7.25 (m, 8H), 7.67 to 7.69 (d, 2H).

N- (phenyl-N- (p-toluenesulfonamido) acridine-9-carboxamide

N- (phenyl) -p-toluenesulfonamide (247 mg, 1 mmol) was dissolved in toluene and treated under argon with potassium tert-butoxide (112 mg, 1 mmol). After the solution was stirred for 30 minutes, the solvent was removed under reduced pressure to give a white solid. The potassium salt was resuspended in anhydrous tetrahydrofuran under argon and a solution of acridine-9-carboxylic acid chloride [by reflux of acridine-9-carboxylic acid (156 mg, 0.75 mmol) and thionyl chloride (3 ml)] in methylene chloride was obtained , added. Triethylamine was added and the reaction mixture was stirred at room temperature overnight. After evaporation of the solvent, the residue was redissolved in ethyl acetate and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give a crude material which was chromatographed on silica gel (30% ethyl acetate / hexane) to give the pure product as an off-white solid.
¹ N NMR _(CDCl3) δ 2.57 (s, 3H), 6.85 to 7.03 (m, 5H), 7.51 to 8.09 (m, 12H).

10-methyl-N- (phenyl-N- (p-toluenesulfonamido) acridinium-9-carboxamidtrifluormethansulfonat

N- (phenyl) -N- (p-toluenesulfonamido) acridine-9-carboxamide (30 mg, 0.0068 mmol) was dissolved in methylene chloride (5 mL) under argon and methyl trifluoromethanesulfonate (77 μL, 0.068 mmol) was added , The solution was stirred overnight at room temperature to give a yellow precipitate. Hexane was added and the precipitate was filtered. The solid was also washed with ether and dried to give the product as yellow crystals.
¹ N NMR (acetone-d ₆ ) δ 2.58 (s, 3H), 5.02 (s, 3H), 7.02-8.79 (m, 12H).

10-methyl-N- (phenyl) -N- (p-toluenesulfonamido) acridan-9-carboxamide (1e)

10-Methyl-N- (phenyl) -N- (p-toluenesulfonamido) acridinium-9-carboxamide trifluoromethanesulfonate (10 mg, 0.0016 mmol) was suspended in absolute ethanol (10 mL) and the solution was heated at reflux for 15 minutes , whereby a clear solution was obtained. Ammonium chloride (88 mg, 1.6 mmol) was added to the solution in portions followed by zinc (108 mg, 1.6 mmol). The yellow color of the solution disappeared immediately after the addition of zinc. The colorless solution was refluxed for 2 hours. The TLC of the reaction mixture showed complete conversion to a non-polar material. The solution was filtered and the precipitate was washed with ethanol (3 x 20 ml). The solution was concentrated to give an off-white solid which was redissolved in methylene chloride and washed with water (2 x 15 mL). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative TLC using 30% ethyl acetate: hexane. The pure product was obtained as an off-white solid.
¹ H NMR _(CDCl3) δ 2.40 (s, 3H), 3.18 (s, 3H), 5.00 (s, 1H), 6.76 to 7.77 (m, 12H).

Measurements of chemiluminescence

The experiments in the following examples done with a luminometer Turner Designs TD-20e, which with a Neutral filter for the light attenuation was connected. The collection of data, analysis and display were controlled by the software. A constant temperature was through maintained an externally circulating water bath, with the Luminometer was connected.

6. Comparison of the compounds 1a-e at pH = 8 9 time course and total intensity

A 200 μl volume of formulation containing 0.01 mM of acridan compound 1a-e in 0.1 M phosphate buffer, pH 8.9, 0.015% (6.2 mM) N ₂ O ₂ , 2.25 mM p Iodophenol, 0.5% (w / w) Tween 20 and 1 mM EDTA was reacted with 1 x 10 ^-3 moles of horseradish peroxidase. 1 shows a comparison of the light emission curves under these conditions. In the following, the maximum value of the light intensity (I _max ) in relative light units (RLU), the time to the maximum light intensity (t _max ) and the total emitted light are compared.

• 1a. Phenyl-l0-methylacridan-9-carboxylate.
• 1b. 4'-hydroxyphenyl-l0-methylacridan-9-carboxylate.
• 1c. 3'-Hydroxyphenyl-l0-methylacridan-9-carboxylate.
• 1d. 6'-hydroxynaphthyl-l0-methylacridan-9-carboxylate.
• 1e. N- (phenyl) -N- (p-toluenesulfonamido) -10-methylacridan-9-carboxamide. The Compound 1b can for different assay purposes best. The p-position of the OH group elevated the amount of light clearly.

7. Sensitivity of detection of horseradish peroxidase with 1b and hydrogen peroxide

An attempt to optimize a matrix was made with 1b (0.1 to 0.05 mM), hydrogen peroxide (4.4 mM to 44 μM), HRP (9 x 10 ^{-1 9} to 1.4 x 10 ^-12 mol) 37 ° C performed. The final assay reagent consisted of 2.25 x 10 ³ M p-iodophenol, 0.5% Tween 20 and 1 x 10 ^-3 M EDTA in 0.1 M Tris buffer pH 8.0. The best compromise between sensitivity and dynamic range was achieved with 1b (46 μmol / L) and 0.2 mmol / L hydrogen peroxide. These conditions gave a linear assay for HRP in the range from 9 × 10 ^{- 19} to 1.4 × 10 ^-14 mol (detection limit S / B = 1.4 after 5 min at 9 × 10 ^-19 mol) or in the range of 9 × 10 ^-19 to 1.4 × 10 ^-15 mol (detection limit S / B = 2 after 15 min at 9 × 10 ^-19 mol).

The 2A to 2C show the relationship between background corrected signal and HRP level using solutions containing 0.05 mM b 0.5% Tween 20.1 x 10 ³ M EDTA in 0.1 M Tris buffer pH = 8.0 contained. The incubation times and [H ₂ O ₂ ] are as in the specified.

B. Sensitivity of the detection of horseradish peroxidase with 1b and sodium perborate

An attempt to optimize a matrix was made with 1b (0.1 to 0.05 mM), sodium perborate (3 mM to 0.2 μM) and HRP (1.4 x 10 ^-18 to 1.4 x 10 ^-14 mol). carried out at 37 ° C. The final assay reagent consisted of 2.25 x 10 ^-3 M of p-iodophenol, 0.5% Tween 20 and 1 x ^10-3 M EDTA in 0.1 M Tris buffer with pH = 8.0. The best compromise between sensitivity and dynamic range was achieved with 1b (46 μmol / l) and 0.2 mmol / l perborate. These conditions gave a linear assay for HRP in the range 0.4 x 10 ^-18 to 1.4 × 10 ^-14 mol (detection limit S / B = 1.4 after 5 min at 1.4 × 10 ^{- 18} moles) or in the range of 1.4 x 10 ^{- from 18} to 1.4 x ^10-15 mol (detection limit S / B = 1.5 after 15 min at 1.4 × 10 ^-18 mol).

The 3A to 3C show the relationship between background corrected signal and HRP level using solutions containing 0.05 mM 1b, 0.5% Tween 20.1 x 10 ^-3 M EDTA in 0.1 M Tris buffer containing pH = 8.0. Incubation times and [NaBO ₃ ] are as indicated in the figures.

9. Influence of pH and the buffer salt

The present invention can be carried out in a pH range of at least 7 to 9 and works with various buffering salts. Tubes containing 200 μl of a formulation containing 0.1 mM 1b in the given buffer, 0.8 mM H ₂ O ₂ , 2.25 mM p-iodophenol, 0.5% (w / w) Tween 20 and 1 mM EDTA were added to the luminometer at room temperature. Horseradish peroxidase (1.4 × 10 ^-15 mol) was injected and the intensity of chemiluminescence was determined at 30 min. The time course of the light emission was similar in all four solutions. The optimum pH may change with changes in the concentrations of the reactants. buffer S / B 0.1 M Tris buffer, pH = 8.0 1400 0.1 M Tris buffer, pH = 8.5 600 0.1 M Tris buffer, pH = 8.9 160 0.1 M phosphate buffer, pH = 8.9 306

10. Optimization the concentration of amplifier and peroxide

The enhancement of light emission from the HRP-catalyzed oxidation of 1b was studied with p-iodophenol. A series of concentrations of p-iodophenol (0.23 to 4.5 mM) in 0.1 M Tris buffer, pH = 8.0, was investigated. The signal-to-background ratios obtained after incubating with 7 x 10 ^-6 moles of HRP for 50 minutes were compared using different assay reagents 1b (0.1 to 0.05 mM) and peroxide (0, 2 to 0.8 mM) were used. The best compromise between sensitivity and concentration was achieved with 1.1 mM p-iodophenol at 0.8 mM peroxide and 0.05 mM 1b. At the best level, with p-iodophenol and 7 x 10 ^-6 moles of the enzyme, a 2500-fold increase in chemiluminescence intensity was achieved (compared to an identical solution containing no enhancer).

4 shows the relationship between the background corrected signal and the HRP amount using solutions containing p-iodophenol (0 to 4.5 mM), 0.5% Tween 20.1 x 10 ³ M EDTA in O, 1 M Tris buffer, pH = 8.0 and 7 x 10 ^-16 moles of HRP, after a reaction time of 15 minutes at 37 ° C.

11. improvement detection by phenol enhancer

In 220 μl of a formulation containing 0.1 mM b 2.25 mM enhancer, 0.8 mM H 2 O 2, 0.5% (w / w) Tween 20 and 1 mM EDTA in 0.1 M Tris buffer, pH = 8.5, at 25 ° C, an aliquot containing 1 x 10 ^-15 mol of HRP was injected and chemiluminescence was measured at 30 min. Table II below shows that the use of p-iodophenol produced the highest signal, whereas with 2.25 mM p-phenylphenol and 2-naphthol, slightly lower enhancement factors were observed, whereas with p-hydroxycinnamic acid a very low enhancement was observed at this concentration has been. The absolute and relative gain factors obtained for a given amplifier depend on the concentrations of enhancer, peroxide and enzyme. Separate optimization of the concentration of p-hydroxycinnamic acid, for example, leads to an improvement in the amplification factor. TABLE II Amplifier S / B p-iodophenol 600 p-phenylphenol 200 2-naphthol 138 p-hydroxycinnamic acid 9

12. Improvement of the proof by surface-active medium

It was found that certain surfactants Agents, such as nonionic surfactants, improve the applicability of the present invention by to provide a better signal-to-background ratio. The improvement over minimizing background chemiluminescence without the added Peroxidase, possibly due to a slowing of the auto - oxidative decomposition of the Ester. In one experiment, Tween 20 (0.5 to 1%) reduced background luminescence from a solution of 1b by a factor of 65 compared to a similar solution containing the surfactant Medium was missing. SDS (sodium dodecyl sulfate) reduces background luminescence to similar ones Way, however, is for the use in solutions not preferred, the higher Contain enzyme concentrations.

13. Proof of HRP at 25 and 37 ° C

The present invention can be carried out within a temperature range of at least 25 to 37 ° C. The light intensity versus time curves determined by the treatment of 200 μl of a formulation containing 0.05 mM of compound b 0.2 mM N ₂ O ₂ , 0.5 mM p-iodophenol, 0.5% (w / w). Tween 20 and 1mM EDTA in 0.1M Tris buffer, pH = 8.0, incubated at 25 or 37 ° C, was obtained with 10 μl of a solution containing 7 x 10 --M ^. Contained ¹⁶ moles of horseradish peroxidase are in 5 shown. The intensity of the chemiluminescence reaches the maximum faster at 37 ° C.

14. Comparison of the proof of HRP with luminol and 1b

As in 6 , the linearity of detection of HRP with a reagent composition of the invention was compared to that of a commercial optimized reagent containing luminol. 40 μl of a solution containing 1b (0.05 mM), p-iodophenol (2.25 mM), H ₂ O ₂ (0.2 mM), Tween 20 (0.5%), EDTA (1 mM) in 0.1 M Tris, pH = 8.0 and 40 μl of commercial reagent (Amersham, Arlington Heights, Illinois) were incubated at 37 ° C and reacted with varying amounts of HRP. 6 compares the ratios between corrected signal and background at 5 min The reagent containing 1b has a higher detection sensitivity (compare log (S-B) / B = 0) than the luminol reagent. The measurement of light intensity at 15 minutes shows a further decrease in the detection limit for the reagent containing 1b, whereas the results of the luminol reagent are unchanged.

15. Chemiluminescent Detection of proteins by Western Blot

To the sensitivity of reagents of the invention in the detection of proteins by Western blotting, a model system of transferrin was applied to polypeptide bands in to deliver known quantities.

A rabbit anti-goat IgG peroxidase conjugate and rabbit anti-goat IgG peroxidase were obtained from Cappel Products (Durham, NC). Human transferrin and fractionated goat Aniti human transferrin serum were supplied by Sigma Chemical Co. (St. Louis, MO). The IgG sample was centrifuged at 10,000 g for 2 minutes and the supernatant was used in the immunological reaction. The transfer membrane Immobilon ^® -P was from Millipore Corp. (Bedford, MA). In the assay process, X-OMAT AR and OMC films from Kodak (Rochester, NY) were used.

SDS-PAGE was performed with the buffer system described in Laemmli (UK Laemmli, Nature, (London), 227, 680 (1970)). The collection gel was 4.38% acrylamide: 0.12% bisacrylamide. The release gel was 6.81% acrylamide: 0.19% bisacrylamide. After electrophoresis, the gel became 7 to 8 minutes with the transfer buffer containing 20 mM Tris, 153 mM glycine and 20% (v / v) methanol. The gel, which was placed between a layer of the transfer membrane and a layer of 3MM (Whatman) chromatography paper, was placed in the transfer unit (Bio-Rad Laboratories, Richmond, CA). The proteins in the gel were electrochemically eluted for 50 to 60 minutes at 4 ° C. and a constant voltage of 100 V. Then the membrane was placed at 4 ° C in a 50 mM Tris-HCl buffered saline pH 7.4 (TBS) overnight. After this period, the membrane became 15 Washed for a few minutes with TBS.

The membrane was left at room temperature for 1 hour with 0.05% Tween-20 in 50 mM Tris-HCl buffered saline pH = 7.4 (T-TBS) treated with 1% fat-free milk powder (NFM) contained. This blocked membrane was left for 75 minutes at room temperature with a primary antibody (Dilution 1: 500 goat anti-human transferrin IgG fraction) incubated using T-TBS containing 1% NFM.

Thereafter, the membrane was rinsed and washed at room temperature three times for 10 minutes each with T-TBS. The washed membrane was incubated for 1 hour at room temperature with a second antibody (dilution : 25,000 of the rabbit anti-goat IgG peroxidase conjugate) using T-TBS containing 1% NFM. The membrane was rinsed and washed four times for 10 minutes each with T-TBS, followed by washing with TBS for 10 minutes. The washed membrane was soaked in a solution of the detection reagent containing a peroxide compound and 4'-hydroxyphenyl-10-methylacridan-9-carboxylate (1b) for 10 minutes and was dropped between layers of a transparent film. The X-ray film became the membrane 1 to 10 Minutes exposed and developed. Composition of the solution of the detection reagent: Tris buffer, pH = 8.8 0.1M 1b 5 × 10 ^-5 M p-iodophenol 1.1 × 10 ^-3 M Tween 20 0.5% (w / w) NaBO ₃ · 4N ₂ O 1.6 × 10 ^-3 M EDTA 5 × 10 ^-4 M

The transferrin standards used were after a 7 second exposure of the X-ray film Kodak X-OMAT AR ( 7A ) or after the 30-second exposure of the roentgen film OMC ( 7B ) clearly up to 20 pg / field visible.

While In the first hour, different exposures could be made with the membrane be made because the membrane emitted light constantly.

A clear advantage of the detection reagents for HRP conjugates on membranes, the N-alkylacridancarboxylic derivatives included is the longer one Duration of light emission. In the present example, the chemiluminescence at least 3 hours with an x-ray film be detected, thereby optimizing the exposure a lot becomes easy. The emission of chemiluminescence can be up to several hours extended when the concentration of acridan 1b is increased. In contrast, produces the best commercial chemiluminescent reagent for detection of HRP on a membrane only for about 1 hour a sufficient signal.

The previous description is intended only illustrate the present invention, and the present invention is limited only by the following claims.

Claims (42)

Acridan of the formula

wherein R1 is selected from alkyl, heteroalkyl and aralkyl groups and R ₃ and R ₄ are selected from aryl, alkyl, heteroalkyl and aralkyl groups. An acridan according to claim 1, wherein R ₃ and R ₄ are selected from phenyl and naphthyl groups. Acridan according to claim 1 of the formula

A method of producing chemiluminescence which comprises reacting a peroxide compound and a peroxidase with an acridan of the formula

wherein R1 is selected from alkyl, heteroalkyl and aralkyl groups, R5 and R6 are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which promotes the generation of light from the acridan by the reaction with a peroxide and a peroxidase. The process of claim 4 wherein Y is an R ₂ -oxy group (R ₂ -O) and R _{2 is selected} from substituted and unsubstituted aryl groups. The method of claim 5, wherein the group R _{2 is selected} from substituted and unsubstituted phenyl and naphthyl groups. The method of claim 6, wherein the group R _{2 is selected} from hydroxyphenyl and hydroxynaphthyl groups. The method of claim 4, wherein the acridan has the formula:

wherein R ₃ and R _{4 are selected} from aryl, alkyl, heteroalkyl and aralkyl groups. The method of claim 8, wherein R ₃ and R ₄ are selected from phenyl and naphthyl groups. The method of claim 4, wherein the acridan is selected from the group consisting of:

A reagent composition which produces light in the presence of a peroxidase and comprises: (a) an acridan of the formula:

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which facilitates the generation of light from the acridan by the Permits permeation with a peroxide and a peroxidase; (b) optionally, a phenolic compound that enhances light production from the acridan; (c) a peroxide compound involved in the conversion of the acridan with the peroxidase; (d) a chelating agent which prevents the peroxide compound from reacting before the peroxidase is added to this composition; and (e) a nonionic surfactant. A reagent composition according to claim 11, wherein Y is an R ₂ oxy group (R ₂ -O) and R _{2 is selected} from substituted and unsubstituted aryl groups. A reagent composition according to claim 11, wherein the group R _{2 is selected} from substituted and unsubstituted phenyl and naphthyl groups. A reagent composition according to claim 11, wherein the group R _{2 is selected} from hydroxyphenyl and hydroxynaphthyl groups. The reagent composition of claim 11, wherein the acridan has the formula:

wherein R ₃ and R _{4 are selected} from aryl, alkyl, heteroalkyl and aralkyl groups. The reagent composition of claim 15, wherein R ₃ and R ₄ are selected from phenyl and naphthyl groups. The reagent composition of claim 11, wherein the acridan has a formula selected from the group consisting of

A reagent composition according to any one of claims 11 to 17, wherein the chelating agent is a salt of ethylenediaminetetraacetic acid (EDTA). A reagent composition according to claim 18, wherein the phenolic compound selected from the group is composed of p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, 2-cyano-6-hydroxybenzothiazole and 2-naphthol. A reagent composition according to any one of claims 11 to 19, wherein the molar ratio between the Phenol compound and the acridan is between about 0.001 and 100. Method according to one of claims 4 to 10, which is the detection an analyte in a chemiluminescent assay method Reaction serves, and in which the acridan with the peroxide and the peroxidase which generates light to detect the analyte. The method of claim 21, wherein the peroxidase is a reagent composition according to one of the claims 11 to 20 is added so that light for the Detection of the analyte is generated. The method of claim 21 or claim 22, wherein the enzyme Peroxidase is coupled with a compound that is specific to the analyte binds. The method of claim 23, wherein the analyte-binding compound, to which the enzyme peroxidase is coupled, selected from the group made of antibodies, Oligonucleotides, haptens and proteins. A method according to any one of claims 21 to 24, wherein the detection done on a membrane. The method of claim 25, wherein the membrane is selected from the group selected is made of nitrocellulose, nylon and polyvinylidene difluoride membranes consists. A method according to any one of claims 21 to 26, wherein the generated Chemiluminescence is detected on a photographic film. A method according to any one of claims 21 to 26, wherein the generated Chemiluminescence is detected with a luminometer. The method of claim 21 or claim 22, wherein the analyte Is hydrogen peroxide. The method of claim 29, wherein the peroxidase is selected from the group selected is made up of horseradish peroxidase, microperoxidase and lactoperoxidase consists. The method of claim 21 or claim 22, wherein the analyte the enzyme is peroxidase. The method of claim 30, wherein the peroxide is selected from the group selected is, which consists of hydrogen peroxide and perborate salts. A kit for detecting an analyte in an assay by a chemiluminescent reaction to produce light having in separate containers: (a) an acridan of the formula:

wherein R _{1 is selected} from alkyl, heteroalkyl and aralkyl groups, R ₅ and R ₆ are selected from the group consisting of hydrogen and non-interfering substituents, and Y is a leaving group which facilitates the generation of light from the acridan by the Permits permeation with a peroxide and a peroxidase; (b) the enzyme peroxidase, wherein the light is detected in the assay method by the reaction of the reagent composition with the peroxidase. The kit of claim 33, wherein Y is an R ₂ -oxy group (R ₂ -O) and R _{2 is selected} from substituted and unsubstituted aryl groups. The kit of claim 34, wherein the group R _{2 is selected} from substituted and unsubstituted phenyl and naphthyl groups. The kit of claim 34, wherein the group R _{2 is selected} from hydroxyphenyl and hydroxynaphthyl groups. The kit of claim 33, wherein the acridan has the formula:

wherein R ₃ and R _{4 are selected} from aryl, alkyl, heteroalkyl and aralkyl groups. 38. The kit of claim 33, wherein R ₃ and R ₄ are selected from phenyl and naphthyl groups. The kit of claim 33, wherein the acridan has a formula selected from the group consisting of the formulas:

Kit according to one of the claims 33 to 39, wherein the acridan is in a reagent composition, their components may be in a single or multiple containers can, optionally a phenolic compound that produces light amplified from the acridan, a peroxide compound which is involved in the conversion of the acridan with the Peroxidase is involved, a chelating agent that prevents the peroxide compound reacts before the peroxidase is added to the composition, and a nonionic surfactant Having means. The kit of claim 40, wherein the peroxide compound is selected from the group selected is, which consists of hydrogen peroxide and perborate salts. Kit according to one of the claims 33 to 41, wherein the enzyme peroxidase from horseradish peroxidase, microperoxidase and lactoperoxidase is.